Substituted tetracycline compounds for treatment of bacillus anthracis infections

ABSTRACT

Methods and compositions for the treatment of  Bacillus anthracis  infections are described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/851,211, filed on Oct. 11, 2006, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

In the fall of 2001, letters intentionally contaminated with Bacillusanthracis were mailed to individuals in Florida, Washington, DC, and NewYork City. These events resulted in exposures both at the sites ofdelivery and also at sites the letters passed through in New Jersey,Pennsylvania, Virginia, Maryland, and Connecticut. In total, there were11 cases of documented inhalation anthrax infections, including 5deaths, and 11 cases of documented cutaneous anthrax infections.Antimicrobial prophylaxis for at least 60 days was recommended for about10,000 individuals; ultimately, about 32,000 people actually receivedprophylactic therapy.

The public health crisis in antibiotic resistance generally focuses onnosocomial and community-acquired infections with organisms that havenaturally become resistant to multiple agents. This situation hasdeveloped due to a combination of antibiotic use (including overuse andmisuse) and the emergence of freely transmissible resistancedeterminant(s). Organisms that might be (or have been) used bybioterrorists could acquire antibiotic resistance not only naturally,but also as a result of intentional manipulation.

Ciprofloxacin, doxycycline, and penicillin G procaine (penicillin) arethe three drugs currently approved for intravenous therapy of all formsof anthrax (cutaneous (skin), inhalation, and gastrointestinal)infection. Mobile elements that confer resistance to tetracyclines andpenicillins can be introduced into B. anthracis and are functional;resistance to ciprofloxacin can be induced by passage in vitro. Thus,there is a real possibility of multiple drug resistant (MDR) anthrax andalternative agents effective against such strains are needed.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains to novel, narrow-spectrum,orally bioavailable substituted tetracycline compounds that are activeagainst B. anthracis, including strains expressing resistance to knowntetracycline resistance elements.

In a further embodiment, the invention pertains to a method for treatinga Bacillus anthracis infection in a subject. The method includesadministering to the subject an effective amount of a substitutedtetracycline compound, such that the Bacillus anthracis infection in thesubject is treated.

In another embodiment, the invention also pertains to a pharmaceuticalcomposition comprising an effective amount of a substituted tetracyclinecompound for the treatment of a Bacillus anthracis infection and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention pertains to a method for treating aBacillus anthracis infection in a subject. The method includesadministering to the subject an effective amount of a substitutedtetracycline compound, such that the Bacillus anthracis infection in thesubject is treated.

The term “Bacillus anthracis infection” includes any state, diseases, ordisorders caused or which result from exposure or alleged exposure toBacillus anthracis or another member of the Bacillus cereus group ofbacteria.

The Bacillus cereus group of bacteria is composed of B. anthracis (theetiologic agent of anthrax), B. cereus and B. weihenstephanensis (foodborne pathogens), B. thuringiensis (an insect pathogen), and B. mycoides(non-pathogenic). B. anthracis is associated with three differentclinical forms of infection. Inhalation anthrax is rare, with only 18cases reported in the US from 1900-1976 and none from 1976-2001. Themortality rate of inhalation anthrax has been reported to range from 40%to 89%; however, many cases are from the pre-antibiotic era {Inglesby,2002 #1942}. Patients that died following the accidental disseminationof B. anthracis from a bioweapons facility in Sverdlovsk, Russia in 1976exhibited hemorrhagic thoracic lymphadenitis, hemorrhagic mediastinitis,and pleural effusions. This experience confirmed that typicalbronchopneumonia is not a characteristic of pulmonary anthrax.

The most common infection due to B. anthracis is cutaneous anthrax,which is rarely fatal when treated with appropriate antibiotics.Gastrointestinal anthrax may develop after eating improperly prepared,contaminated meat; these infections are typically encountered indeveloping countries in Africa and Asia.

The term “subject” includes animals (e.g., mammals, e.g., cats, dogs,horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates(e.g., chimpanzees, gorillas, and humans)) which are capable of (orcurrently) suffering from a Bacillus anthracis infection. It alsoincludes transgenic animal models.

The term “treated,” “treating” or “treatment” includes therapeuticand/or prophylactic treatment of a Bacillus anthracis infection. Thetreatment includes the diminishment or alleviation of at least onesymptom associated or caused by a Bacillus anthracis infection. Forexample, treatment can be diminishment of one or several symptoms of aBacillus anthracis infection or complete eradication.

The language “effective amount” of the tetracycline compound is thatamount necessary or sufficient to treat or prevent a Bacillus anthracisinfection in a subject, e.g. prevent the various morphological andsomatic symptoms of multiple sclerosis. The effective amount can varydepending on such factors as the size and weight of the subject, thetype of illness, or the particular tetracycline compound. For example,the choice of the tetracycline compound can affect what constitutes an“effective amount.” One of ordinary skill in the art would be able tostudy the aforementioned factors and make the determination regardingthe effective amount of the tetracycline compound without undueexperimentation.

The term “tetracycline compound” does not include minocycline,doxycycline, or tetracycline. The term includes substituted tetracyclinecompounds or compounds with a similar ring structure to tetracycline.Examples of tetracycline compounds include: chlortetracycline,oxytetracycline, demeclocycline, methacycline, sancycline, chelocardin,rolitetracycline, lymecycline, apicycline; clomocycline, guamecycline,meglucycline, mepylcycline, penimepicycline, pipacycline, etamocycline,penimocycline, etc. Other derivatives and analogues comprising a similarfour ring structure are also included (See Rogalski, “ChemicalModifications of Tetracyclines,” the entire contents of which are herebyincorporated herein by reference). Table 1 depicts tetracycline andseveral known other tetracycline derivatives.

TABLE 1

Other tetracycline compounds which may be modified using the methods ofthe invention include, but are not limited to,6-demethyl-6-deoxy-4-dedimethylaminotetracycline; tetracyclino-pyrazole;7-chloro-4-dedimethylaminotetracycline;4-hydroxy-4-dedimethylaminotetracycline;12α-deoxy-4-dedimethylaminotetracycline;5-hydroxy-6α-deoxy-4-dedimethylaminotetracycline; 4-dedimethylamino-12α-deoxyanhydrotetracycline;7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline 4,6-hemiketal;4-oxo-11a C1-4-dedimethylaminotetracycline-4,6-hemiketal;5a,6-anhydro-4-hydrazon-4-dedimethylamino tetracycline;4-hydroxyimino-4-dedimethylamino tetracyclines;4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;4-amino-4-dedimethylamino-5a,6 anhydrotetracycline;4-methylamino-4-dedimethylamino tetracycline;4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline; tetracycline quaternary ammonium compounds;anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides; 4-ketotetracyclines; 5-keto tetracyclines; 5a, 11a dehydro tetracyclines; 11aC1-6,12 hemiketal tetracyclines; 11a C1-6-methylene tetracyclines; 6,13diol tetracyclines; 6-benzylthiomethylene tetracyclines; 7,11adichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro(α)-6-demethyl-6-deoxy tetracyclines; 6-fluoro (β)-6-demethyl-6-deoxytetracyclines; 6-α acetoxy-6-demethyl tetracyclines;6-βacetoxy-6-demethyl tetracyclines; 7,13-epithiotetracyclines;oxytetracyclines; pyrazolotetracyclines; 11a halogens of tetracyclines;12a formyl and other esters of tetracyclines; 5,12a esters oftetracyclines; 10,12a-diesters of tetracyclines; isotetracycline;12-a-deoxyanhydro tetracyclines;6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines; B-nortetracyclines;7-methoxy-6-demethyl-6-deoxytetracyclines;6-demethyl-6-deoxy-5a-epitetracyclines; 8-hydroxy-6-demethyl-6-deoxytetracyclines; monardene; chromocycline; 5a methyl-6-demethyl-6-deoxytetracyclines; 6-oxa tetracyclines, and 6 thia tetracyclines.

The term “substituted tetracycline compound” includes tetracyclinecompounds with one or more additional substituents, e.g., at the 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 12a or 13 position or at any otherposition which allows the substituted tetracycline compound of theinvention to perform its intended function, e.g., treat B. anthracisinfections.

In a further embodiment, the substituted tetracycline compound has anMIC less than that of doxycycline for at least one strain of Bacillusanthracis. The MIC of the substituted tetracycline compound can betested using the method described in the Examples. In a furtherembodiment, the substituted tetracycline compound has an MIC less than32 μg/ml for a doxycycline resistant strain of Bacillus anthracis. In afurther embodiment, the MIC of the substituted tetracycline has an MICthat is 90% or less, 50% or less, 20% or less, 10% or less, 5% or lessthan the MIC of doxycycline for a particular strain of Bacillusanthracis.

In a further embodiment, the substituted tetracycline compound has anMIC less than that of ciproflaxin for at least one strain of Bacillusanthracis. The MIC of the substituted tetracycline compound can betested using the method described in the Examples. In a furtherembodiment, the substituted tetracycline compound has an MIC less than32 μg/ml for a ciproflaxin resistant strain of Bacillus anthracis. In afurther embodiment, the MIC of the substituted tetracycline has an MICthat is 90% or less, 50% or less, 20% or less, 10% or less, 5% or lessthan the MIC of ciproflaxin for a particular strain of Bacillusanthracis.

In a further embodiment, the substituted tetracycline compound of theinvention is of the formula I:

wherein

R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen, optionally linkedto R² to form a ring;

R^(2″) is cyano or C(═O)—NR²R^(2′);

R² is hydrogen, alkyl, halogen, alkenyl, alkynyl, aryl, hydroxyl, thiol,cyano, nitro, acyl, formyl, alkoxy, amino, alkylamino, heterocyclic, orabsent, optionally linked to R¹ to form a ring;

R^(2′), R³, R^(4a), and R^(4b) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R¹⁰, R¹¹, and R¹² are each independently hydrogen, alkyl, aryl, benzyl,arylalkyl, or a pro-drug moiety;

R⁴ and R^(4′) are each independently NR^(4a)R^(4b), alkyl, acyl,alkenyl, alkynyl, hydroxyl, halogen, hydrogen, or taken together═N—OR^(4a);

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, dialkylamino, hydroxyl, halogen, thiol, nitro, alkyl,alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, boronic ester, alkylcarbonyl, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃ (NR^(8c))₀₋₁C(=E′)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkylheterocyclic, thionitroso, or —(CH₂)₀₋₃ (NR^(9c))₀₋₁ C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d), R^(9e), andR^(9f) are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR^(8f), or S;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f), or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f), and pharmaceutically acceptable salts, estersand enantiomers thereof.

In a further embodiment, R² is C(═O)NH₂; R³, R¹⁰, R¹¹, and R¹² are eachhydrogen or a prodrug moiety; R⁴ is NR^(4a)R^(4b); R^(4a) and R^(4b) areeach methyl; R⁵ is hydrogen; R⁸ is hydrogen; X is CR⁶R^(6′); R⁶ ishydrogen; and R^(5′) and R^(6′) are hydrogen.

In another further embodiment, R⁸ and R⁹ are hydrogen.

In yet another further embodiment, R⁷ is substituted phenyl, a boronicester, alkylcarbonyl, heterocyclic, aminoalkyl, or arylalkynyl. Examplesof substituents for phenyl R⁷ groups include, but are not limited to,alkoxy, alkyl-O—N═C—CR^(7g)R^(7h), alkylaminoalkyl, alkenylaminoalkyl,alkoxyalkylaminoalkyl, substituted alkyl, and substituted carbonylamino,wherein R^(7g) and R^(9h) are each independently hydrogen or alkyl.

In another further embodiment, R⁷ is substituted or unsubstitutedheteroaryl, e.g., substituted or unsubstituted pyrimidinyl, pyridinyl,or furanyl.

In another further embodiment, R⁷ is substituted or unsubstitutedpiperdinyl-alkyl. In other embodiments, R⁷ is pyridinyl-alkynyl orsubstituted or unsubstituted phenyl-alkynyl.

In other embodiment, R⁷ is hydrogen and R⁹ is substituted carbonylamino.

In other embodiments, R⁸ is hydrogen; R⁷ is heterocyclic, alkyl,alkyl-O—N═C—CR^(7g)R^(7h), or dimethylamino, wherein R^(7g) and R^(9h)are each independently hydrogen or alkyl.

In a further embodiment, R⁹ is aminoalkyl. Examples of aminoalkyl R⁹moieties include aminomethyl moieties and moieties of the formula:

wherein:

J⁵ and J⁶ are each independently hydrogen, alkyl, alkenyl, alkynyl,aryl, sulfonyl, acyl, alkoxycarbonyl, alkaminocarbonyl,alkaminothiocarbonyl, substituted thiocarbonyl, substituted carbonyl,alkoxythiocarbonyl, or linked to form a ring; and

J⁷ and J⁸ are each alkyl, halogen, or hydrogen.

In a further embodiment, J⁷ and J⁸ are each hydrogen.

In another further embodiment, J⁶ is hydrogen and J⁵ is substituted orunsubstituted alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl,2-methyl-propyl, hexyl, and/or cyclohexyl. Examples of substituents ofJ⁵ include one or more fluorines or substituted or unsubstituted phenylgroups.

In another embodiment, J⁵ and/or J⁶ is substituted or unsubstitutedalkyl or alkenyl. Examples of J⁵ and/or J⁶ include methyl, ethyl,propyl, propenyl, 2-methyl-propyl, butyl, butenyl, pentyl, pentenyl,hexyl, and hexenyl. In a further embodiment, J⁵ is substituted with oneor more fluorines or substituted or unsubstituted phenyl groups.

In another further embodiment, J⁵ and J⁶ are linked to form a ring,e.g., a piperdinyl ring or a fused ring, e.g., 2,3-dihydro-indole or andecahydro-isoquinoline. In another further embodiment, the piperidinylring is substituted with one or more halogens, one or more heterocyclicgroups or one or more halogenated alkyl groups (e.g., trifluoromethyl).

In one embodiment, R^(2″) is C(═O)NH₂; R^(4′), R^(5′), R³, R¹⁰, R¹¹, andR¹² are each hydrogen or a prodrug moiety; R⁴ is NR^(4a)R^(4b); R^(4a)and R^(4b) are each methyl; R⁵ is hydroxyl; R⁸ is hydrogen; X isCR⁶R^(6′); R⁶ is hydrogen and R^(6′) is alkyl (e.g., methyl).

In a further embodiment, R⁷ is hydrogen and R⁹ aminoalkyl (e.g.,piperidinyl alkyl, such as halogenated alkyl substituted piperidinylalkyl, for example, trifluoromethyl substituted piperidinylalkyl).

In another embodiment, the substituted tetracycline compound is selectedfrom the group consisting of:

and pharmaceutically acceptable salts thereof.

The tetracycline compounds of this invention can be synthesized usingthe methods described in the Schemes and/or by other techniques known tothose of ordinary skill in the art.

The substituted tetracycline compounds of the invention can besynthesized using the methods described in the following schemes and byusing art recognized techniques. All novel substituted tetracyclinecompounds described herein are included in the invention as compounds.

9- and 7-substituted tetracyclines can be synthesized by the methodshown in Scheme 1. As shown in Scheme 1, 9- and 7-substitutedtetracycline compounds can be synthesized by treating a tetracyclinecompound (e.g., doxycycline, 1A), with sulfuric acid and sodium nitrate.The resulting product is a mixture of the 7-nitro and 9-nitro isomers(1B and 1C, respectively). The 7-nitro (1B) and 9-nitro (1C) derivativesare treated by hydrogenation using hydrogen gas and a platinum catalystto yield amines 1D and 1E. The isomers are separated at this time byconventional methods. To synthesize 7- or 9-substituted alkenylderivatives, the 7- or 9-amino tetracycline compound (1E and 1F,respectively) is treated with HONO, to yield the diazonium salt (1G and1H). The salt (1G and 1H) is treated with an appropriate reactivereagent to yield the desired compound (e.g., in Scheme 1,7-cyclopent-1-enyl doxycycline (1H) and 9-cyclopent-1-enyl doxycycline(1I)).

As shown in Scheme 2, tetracycline compounds of the invention wherein R⁷is a carbamate or a urea derivative can be synthesized using thefollowing protocol. Sancycline (2A) is treated with NaNO₂ under acidicconditions forming 7-nitro sancycline (2B) in a mixture of positionalisomers. 7-nitrosancycline (2B) is then treated with H₂ gas and aplatinum catalyst to form the 7-amino sancycline derivative (2C). Toform the urea derivative (2E), isocyanate (2D) is reacted with the7-amino sancycline derivative (2C). To form the carbamate (2G), theappropriate acid chloride ester (2F) is reacted with 2C.

As shown in Scheme 3, tetracycline compounds of the invention, whereinR⁷ is a heterocyclic (i.e., thiazole) substituted amino group can besynthesized using the above protocol. 7-amino sancycline (3A) is reactedwith Fmoc-isothiocyanate (3B) to produce the protected thiourea (3C).The protected thiourea (3C) is then deprotected yielding the activesancycline thiourea (3D) compound. The sancycline thiourea (3D) isreacted with an α-haloketone (3E) to produce a thiazole substituted7-amino sancycline (3F).

7-alkenyl tetracycline compounds, such as 7-alkynyl sancycline (4A) and7-alkenyl sancycline (4B), can be hydrogenated to form 7-alkylsubstituted tetracycline compounds (e.g., 7-alkyl sancycline, 4C).Scheme 4 depicts the selective hydrogenation of the 7-position double ortriple bond, in saturated methanol and hydrochloric acid solution with apalladium/carbon catalyst under pressure, to yield the product.

In Scheme 5, a general synthetic scheme for synthesizing 7-position arylderivatives is shown. A Suzuki coupling of an aryl boronic acid with aniodosancycline compound is shown. An iodo sancycline compound (5B) canbe synthesized from sancycline by treating sancycline (5A) with at leastone equivalent N-iodosuccinimide (NIS) under acidic conditions. Thereaction is quenched, and the resulting 7-iodo sancycline (5B) can thenbe purified using standard techniques known in the art. To form the arylderivative, 7-iodo sancycline (5B) is treated with an aqueous base(e.g., Na₂CO₃) and an appropriate boronic acid (5C) and under an inertatmosphere. The reaction is catalyzed with a palladium catalyst (e.g.,Pd(OAc)₂). The product (5D) can be purified by methods known in the art(such as HPLC). Other 7-aryl, alkenyl, and alkynyl tetracyclinecompounds can be synthesized using similar protocols.

The 7-substituted tetracycline compounds of the invention can also besynthesized using Stille cross couplings. Stille cross couplings can beperformed using an appropriate tin reagent (e.g., R—SnBu₃) and ahalogenated tetracycline compound, (e.g., 7-iodosancycline). The tinreagent and the iodosancycline compound can be treated with a palladiumcatalyst (e.g., Pd(PPh₃)₂Cl₂ or Pd(AsPh₃)₂Cl₂) and, optionally, with anadditional copper salt, e.g., CuI. The resulting compound can then bepurified using techniques known in the art.

The compounds of the invention can also be synthesized using Heck-typecross coupling reactions. As shown in Scheme 6, Heck-typecross-couplings can be performed by suspending a halogenatedtetracycline compound (e.g., 7-iodosancycline, 6A) and an appropriatepalladium or other transition metal catalyst (e.g., Pd(OAc)₂ and CuI) inan appropriate solvent (e.g., degassed acetonitrile). The substrate, areactive alkene (6B) or alkyne (6D), and triethylamine are then addedand the mixture is heated for several hours, before being cooled to roomtemperature. The resulting 7-substituted alkenyl (6C) or 7-substitutedalkynyl (6E) tetracycline compound can then be purified using techniquesknown in the art.

To prepare 7-(2′-chloro-alkenyl)-tetracycline compounds, the appropriate7-(alkynyl)-sancycline (7A) is dissolved in saturated methanol andhydrochloric acid and stirred. The solvent is then removed to yield theproduct (7B).

As depicted in Scheme 8, 5-esters of 9-substituted tetracyclinecompounds can be formed by dissolving the 9-substituted compounds (8A)in strong acid (e.g., HF, methanesulphonic acid, andtrifluoromethanesulfonic acid) and adding the appropriate carboxylicacid to yield the corresponding esters (8B).

As shown in Scheme 9 below, 7 and 9 aminomethyl tetracyclines may besynthesized using reagents such as hydroxymethyl-carbamic acid benzylester.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles,” “heterocycles,” “heteroaryls” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkyl carbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 6 or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in theirring structure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls,” the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls,” the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. It includes substituted acylmoieties. The term “substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl,” “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The termincludes “alkyl amino” which comprises groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups. The term “arylamino” and“diarylamino” include groups wherein the nitrogen is bound to at leastone or two aryl groups, respectively. The term “alkylarylamino,”“alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which isbound to at least one alkyl group and at least one aryl group. The term“alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to anitrogen atom which is also bound to an alkyl group.

The term “amide,” “amido” or “aminocarbonyl” includes compounds ormoieties which contain a nitrogen atom which is bound to the carbon of acarbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl”or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl and arylcarbonylamino groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms “alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,”“arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,”“alkynylcarbonylamino,” and “arylcarbonylamino” are included in term“amide.” Amides also include urea groups (aminocarbonylamino) andcarbamates (oxycarbonylamino).

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom. Thecarbonyl can be further substituted with any moiety which allows thecompounds of the invention to perform its intended function. Forexample, carbonyl moieties may be substituted with alkyls, alkenyls,alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which containa carbonyl include aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group.

The term “ester” includes alkoxycarboxy groups such as methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.The alkyl, alkenyl, or alkynyl groups are as defined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings.” Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amido, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts,” J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters.

It will be noted that the structure of some of the tetracyclinecompounds of this invention includes asymmetric carbon atoms. It is tobe understood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. Furthermore, thestructures and other compounds and moieties discussed in thisapplication also include all tautomers thereof.

In another further embodiment, the substituted tetracycline compound isadministered in combination with a second agent.

The language “in combination with” a second agent includesco-administration of the tetracycline compound, and with the secondagent, administration of the tetracycline compound first, followed bythe second agent and administration of the second agent, followed by thetetracycline compound. The second agent may be any agent which is knownin the art to treat, prevent, or reduce the symptoms of a Bacillusanthracis infection. Furthermore, the second agent may be any agent ofbenefit to the subject when administered in combination with theadministration of an tetracycline compound.

Examples of second agents include antibiotics, such as rifampin,vancomycin, ampicillin, chloramphenicol, imipenem, clindamycin, andclarithromycin.

In another embodiment, the invention pertains to pharmaceuticalcompositions comprising an effective amount of a substitutedtetracycline compound of the invention for the treatment of a Bacillusanthracis infection and a pharmaceutically acceptable carrier.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a Bacillus anthracis infection. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The tetracycline compounds of the invention may be administered alone orin combination with pharmaceutically acceptable carriers or diluents byany of the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays(e.g., aerosols, etc.), creams, salves, suppositories, jellies, gels,pastes, lotions, ointments, aqueous suspensions, injectable solutions,elixirs, syrups, and the like. Such carriers include solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents,etc. Moreover, oral pharmaceutical compositions can be suitablysweetened and/or flavored. In general, the therapeutically-effectivecompounds of this invention are present in such dosage forms atconcentration levels ranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof. The compositions of the invention may beformulated such that the tetracycline compositions are released over aperiod of time after administration.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.,for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being used, the particular compositions formulated, the mode ofapplication, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a substitutedtetracycline of the invention, for the preparation of a medicament. Themedicament may include a pharmaceutically acceptable carrier and thetetracycline compound is an effective amount, e.g., an effective amountto treat a Bacillus anthracis infection.

Exemplification of the Invention: EXAMPLE 1 Antibacterial Activity ofTetracycline Compounds Against Susceptible and (Multiple) AntibioticResistant Organisms

Efflux. The tetracycline efflux proteins, in general, confer resistanceto both tetracycline and doxycycline. S. aureus RN4250 bears a TetKefflux mechanism and is resistant to both agents, but susceptible tominocycline (Table 2). A number of tetracyclines that overcomegram-positive efflux (Table 2) have been identified.

TABLE 2 MICs (μg/ml) of novel TCs against strains with efflux resistancedeterminants. S. aureus S. aureus S. aureus S. aureus RN450 ^(a) RN4250^(b) RN450 ^(a) RN4250 ^(b) Compound MIC (ug/ml) Compound MIC (ug/ml)Doxycycline 0.06 4 S 0.06 0.06 Minocycline 0.25 0.5 T 0.06 0.06Tetracycline 0.06 64 U 0.06 0.06 O 0.06 0.06 V 0.06 0.06 M 0.06 0.06 W0.06 0.06 Q 0.06 0.06 X 0.06 0.06 P 0.06 0.06 Y 0.06 0.06 ^(a.) Wildtype S. aureus. ^(b.) Contains a TetK (efflux) resistance determinant.

Ribosome protection. The ribosome protection determinants, which conferresistance to tetracycline, doxycycline and minocycline, arepredominantly found in gram-positive bacteria and are probably the mostwidespread tetracycline resistance determinant in these organisms. Anumber of tetracycline compounds that can overcome this mechanism ofresistance in a variety of gram-positive bacteria including S. aureus,E. faecium, and S. pneumoniae (Table 3).

TABLE 3 MICs (μg/ml) of tetracycline compounds against strains withribosome protection resistance determinants S. S. E. S. S. aureus aureusfaecium pneumoniae pneumoniae RN450 ^(a) MRSA5 ^(b) 494 ^(c) 157E ^(a)700905 ^(d) Compound MIC (ug/ml) Doxycycline 0.06 4 8 0.06 4 Minocycline0.25 2 16 0.06 8 Tetracycline 0.06 32 64 0.06 32 Z 0.13 0.5 2 0.06ND^(e) AA 1 0.5 1 0.5 1 AB 0.06 1 0.06 0.06 4 AD 0.06 1 2 0.13 0.5 AE0.06 0.5 2 0.13 1 AK 1 2 1 0.5 0.5 ^(a.) Wild type. ^(b.) Methicillinresistant S. aureus; contains TetM (ribosome protection); alsomulti-drug resistant. ^(c.) Contains TetL (efflux) and TetM (ribosomeprotection); is also resistant to vancomycin and erythromycin. ^(d.)Contains TetM (ribosome protection); is also resistant to penicillin anderythromycin. ^(e)ND, not determined.

Efflux and ribosome protection concurrently. A number of tetracyclinecompounds were tested against gram-positive bacteria possessing bothtetracycline efflux and ribosome protection determinants as well asother non-tetracycline resistance mechanisms. Compounds withsubstitutions at both R⁷ and R⁹ position in Formula 1 e.g., substituted7-dimethylamino-9-aminomethylcyclines and 7-aryl or heteroarylsancyclines) demonstrated activity against both tetracycline sensitiveisolates and tetracycline resistant gram-positive bacteria containingefflux and ribosome protection determinants (Table 4).

TABLE 4 MICs (μg/ml) of tetracycline compounds against strains withribosome protection and efflux resistance determinants. E. faecium E.faecalis S. aureus S. pneumoniae 494 ^(a) 29212 ^(b) MRSA5 ^(c) 700905^(d) Compound MIC (ug/ml) Doxycycline 16 4 4 4 Minocycline 16 4 2 8Tetracycline 64 16 32 32 A 1 1 1 0.25 B 1 1 1 0.5 C 0.25 0.5 1 0.25 D 10.25 1 0.25 E 1 0.25 0.25 0.06 F 1 0.5 0.5 0.25 G 1 0.5 0.5 0.06 H 0.50.5 0.35 0.06 I 1 1 1 0.5 J 1 0.25 0.5 0.06 K 1 1 0.5 0.25 L 1 1 0.50.75 R 1 2 1 0.13 N 0.5 1 1 0.13 AH 0.25 0.25 1 0.06 ^(a.) Has TetM(ribosome protection) and TetL (efflux); is resistant to vancomycin anderythromycin. ^(b.) Has TetM (ribosome protection). ^(c.) Methicillinresistant S. aureus; contains TetM, ribosome protection; also multi-drugresistant. ^(d.) Has TetM (ribosome protection).

Bacillus cereus. In order to prevent the unnecessary use of the anthraxpathogen, a group of tetracycline resistant B. cereus was obtained. Inthis panel, B. cereus 95/3032 and 98/2658 were classified astetracycline susceptible whereas B. cereus 98/2620 and 97/4144 weretetracycline resistant (Table 6). Preliminary MICs were determined forcommon antibiotics against the B. cereus isolates (Table 5).

B. cereus containing natural tetracycline resistance determinants werechosen rather than creating isogenic tetracycline resistant B. anthracisstrains since it would be a violation of International Bioweapons Treatyto purposefully create an antibiotic resistant category A agent. Inaddition, B. cereus are generally more tetracycline resistant than B.anthracis.

TABLE 5 Activity of tetracycline compounds against tetracyclinesusceptible and tetracycline resistant Bacillus cereus. B. cereus B.cereus B. cereus B. cereus 98/2620 ^(a) 95/3032 ^(b) 98/2658 ^(c)97/4144 ^(d) Compound MIC (ug/ml) Doxycycline 4 ≦0.06 ≦0.06 4Minocycline 0.5 ≦0.06 ≦0.06 0.5 Tetracycline 32 ≦0.06 ≦0.06 64Cefotaxime 64 >64 >64 32 Penicillin 32 >64 >64 >64 Vancomycin 1 1 2 1Erythromycin ≦0.06 0.125 1 0.125 Clindamycin 0.25 0.5 0.5 0.5 ^(a) . Anindustrial fermenter isolate, serotype 1. ^(b.) Isolated from anorthopedic-related area, serotype 24. ^(c.) Non-typeable. ^(d.) Isolatedfrom an individual with food poisoning, serotype AA.

Bacillus anthracis. The panel of B. anthracis isolates (n=27) that wasavailable for susceptibility studies included two organisms that exhibitreduced susceptibility to doxycycline (Table 6). B. anthracis V770 was4->33-fold less susceptible to doxycycline than 25 other B. anthracisand strain V770NPIR was fully doxycycline-resistant.

The group of organisms listed in Table 6 all possessed the sametetracycline resistance determinants that would be found in B. anthracisand the majority were multi-drug resistant. The criteria for selectingcompounds for subsequent testing in B. anthracis were (a) the compoundsmust not possess cytotoxicity in vitro (Table 9) and (b) the compoundswere required to possess a MIC of ≦0.5 μg/ml against this panel ofresistant isolates (Table 7). At least five tetracycline compounds wereidentified (Table 7).

The activities of these tetracyclines were tested against B. anthracis(n=5), including the tetracycline resistant strains V770 and V770NPIR(Table 7). As illustrated, these compounds possessed exceptionalactivity against tetracycline susceptible and resistant B. anthracisisolates in vitro (Table 7). Compounds AI, H, and AJ all containsubstituents at the R⁹ position of the tetracycline core while compoundsAM and AF bear substitutions at the R⁷ and R⁹ positions. Without beingbound by theory, these data support the hypothesis that tetracyclinecompounds directed against common tetracycline resistant organisms,e.g., S. aureus, S. pneumoniae, and Enterococcus spp. may also targettetracycline resistant B. anthracis.

TABLE 6 Activity of tetracycline compounds against susceptible anddoxycycline resistant B. anthracis. Vollum1B Sterne Ames V770 V770NPIRCompound MIC (ug/ml) Ciprofloxacin 0.25 0.25 0.25 0.12 0.25 Doxycycline^(a) 0.06 0.12 <0.03 1 32 AI <0.03 <0.03 0.06 0.12 0.06 AM 0.06 0.060.06 0.12 0.06 AF <0.03 <0.03 <0.03 0.06 0.06 H <0.03 <0.03 <0.03 <0.030.06 AJ <0.03 <0.03 <0.03 <0.03 <0.03 ^(a.) The activity of doxycyclineagainst the entire B. anthracis panel (n = 27) is as follows: MIC50 =0.06 μg/ml; MIC90 = 0.25 μg/ml; MIC Range = <0.03-32 μg/ml.

TABLE 7 Activity of tetracycline compounds against common susceptibleand tetracycline resistant bacteria. S. aureus E. faecium E. faecalis S.pneumoniae MRSA5 ^(a) 494 ^(b) 29212 ^(c) 700905 ^(d) Compound MIC(ug/ml) Doxycycline 4 16 4 4 Minocycline 2 16 4 8 Tetracycline 32 64 1632 AI 0.5 0.5 0.5 0.06 AM 0.25 0.25 0.13 0.06 AF 0.13 0.25 0.13 0.06 H0.35 0.5 0.5 0.06 AJ 0.5 0.5 0.5 0.06 ^(a.) Methicillin resistant S.aureus; contains TetM, ribosome protection; also multi-drug resistant.^(b.) Has TetM (ribosome protection) and TetL (efflux); is resistant tovancomycin and erythromycin. ^(c.) Has TetM (ribosome protection). ^(d.)Has TetM (ribosome protection); is resistant to penicillin anderythromycin.

EXAMPLE 2 Additional Potential Mechanisms of Antibacterial Activity byTetracycline Compounds

In addition to inhibiting protein synthesis, molecules within thetetracycline family are reported to affect peptidoglycan biosynthesis.Using cell-free macromolecular synthesis assays early studies dividedthe tetracycline compounds into two classes based on these additionalactivities. Class 1 compounds (tetracycline, minocycline, anddoxycycline) were potent inhibitors of protein synthesis compared to theweak effects of class 2 molecules (chelocardin, anhydrotetracycline, and4-epi-anhydrochlorotet).

Using chemical footprinting assays, minocycline, doxycycline, andtetracycline were shown to affect the reactivity of nucleotides known tomediate binding of the antibiotics within the 16S rRNA. Tigecyclineexhibited a chemical footprint similar to that of tetracycline. Asimilar effect was not seen with chelocardin or anhydrotetracycline,which correlates with their poor activity against the purified ribosomein vitro.

As illustrated in Table 8, these previous findings were confirmed andmethods for deriving IC50 values (i.e., compound concentration necessaryto inhibit a biological process by 50%) were developed. In particular,compounds AA, O, and tigecycline have a profile similar to class Icompounds. Compounds AA and A also affect peptidoglycan biosynthesis.

TABLE 8 Effect of tetracycline compounds on macromolecular synthesis ofS. aureus RN450. Protein Peptidoglycan MIC synthesis ^(a) synthesisAntibiotic (μg/ml) IC50 IC90 IC50 IC90 Tetracycline 0.06 <0.030.11 >32 >32 Minocycline 0.19 <0.03 0.1 4.6 20.9 Doxycycline 0.06 <0.03<0.03 3.9 18.23 Anhydro- 2 <0.03 <0.03 19.2 >32 tetracycline Tigecycline2 0.12 0.68 >32 >32 AA 1 0.14 1.4 3.8 23 AB 0.06 0.19 1.8 18.3 >32 A0.25 1.9 5 2.1 3.8 AE 0.06 0.07 0.62 6.0 >32 O 0.06 0.33 0.92 >32 >32^(a.) Compounds were assayed against S. aureus RN450, a TC susceptibleorganism and IC50 and IC90 values are reported in μg/ml.

EXAMPLE 3 In vitro and In vivo Toxicity

An in vitro determination of the cytotoxicity of the compounds of theinvention was performed using standard mammalian cell assays and in vivousing mice. Specifically, African green monkey kidney (COS-1) andChinese hamster ovary (CHO-K1) cell lines were used according to tostandard methods (see Zhi-Jun, Y., N. Sriranganathan, T. Vaught, S. K.Arastu, and S. A. Ahmed. 1997. A dye-based lymphocyte proliferationassay that permits multiple immunological analyses: mRNA, cytogenetic,apoptosis, and immunophenotyping studies. J Immunol Methods 210:25-39).Briefly, suspensions of tissue culture cells were grown overnight in thepresence of serial dilutions of drug up to a maximum concentration of 50or 100 μg/ml. The metabolism of the tissue culture cells was monitoredwith resazurin, a soluble non-toxic dye. Control cytotoxic andnon-cytotoxic compounds were routinely included in all assays. Tox100values represented the concentration of compound necessary to inhibitcellular proliferation by 100%. Compounds without measurablecytotoxicity in vitro were assigned a Tox100 value of greater than thehighest concentration assayed (e.g., 50 or 100 μg/ml). The results areshown in Table 9.

TABLE 9 Cytotoxicity of tetracycline compounds. Tox50 (μg/ml) ^(a)Compound COS-1 CHO-K1 AI >100 >100 AM >100 92.85 AF >100 >100H >100 >100 AJ >100 >100 ^(a.) Represents the concentration necessary tocause 50% inhibition of cell growth in tissue culture cells.

EXAMPLE 4 Efficacy of Tetracycline Compounds Against Susceptible andResistant Organisms in Animal Infection Models

In vivo efficacy as well as oral bioavailability of the tetracyclinecompounds were assessed in murine models of infection and compared tocontrol tetracyclines and other currently available antibiotics. In thestandard screening assay of acute systemic infection (Table 10), micewere given a lethal intraperitoneal inoculum of S. pneumoniae strain157E (tetracycline susceptible) or 700905 (tetracycline resistant),followed by a single dose of drug, and then observed for survival over48 hours. Each experiment routinely included an untreated group (n=5;expected survival <5%) and a group (n=5) treated with a conventionalantibiotic (e.g., minocycline, ciprofloxacin, and ampicillin; expectedsurvival >80%). The results are tabulated in Table 10.

TABLE 10 Efficacy of selected tetracyclines in the screening assay ofacute systemic infection due to S. pneumoniae 157E. SC PO Compound dose% survival dose % survival B 5 mg/kg 100%  5 mg/kg  40% C 5 mg/kg 100%10 mg/kg  60% D 5 mg/kg  0% 10 mg/kg  0% AI 5 mg/kg  40% 10 mg/kg  0% E5 mg/kg  40% 10 mg/kg  0% AM 5 mg/kg 100% 50 mg/kg  0% F 5 mg/kg 100% 50mg/kg 100% AJ 5 mg/kg 100% 50 mg/kg  80% ND, not determined.

Compounds providing ≧60% survival at 10 mg/kg were further assessed in adose response study to determine the PD50 (the drug concentrationnecessary to prevent death in 50% of the mice in a treatment group).These experiments involved an untreated group, a group treated with acontrol antibiotic (e.g., minocycline, ciprofloxacin, and ampicillin),and up to five groups each receiving a different doses of an activeexperimental compound; all groups included 5 animals (Table 11).Compounds B and C were efficacious following tetracycline administrationand compounds H and I exhibited oral activity (Tables 10 and 11).Compound H, which exhibited potency against tetracycline resistant B.anthracis (Table 6), exhibits IV and PO efficacy against infectionscaused by tetracycline susceptible and resistant organisms (Table 11),and is efficacious in a model of lung infection following IV and PO drugadministration (Table 12).

TABLE 11 Efficacy (PD50) of selected tetracycline compounds in mice withacute systemic infection due to S. pneumoniae. S. pneumoniae 157E S.pneumoniae 700905 IV PO IV PO Compound PD50 (mg/kg) Minocycline 0.531.5 >50 >50 Ciprofloxacin >50 ND ND >50 Ampicillin 0.6 1.1 43.7 >50 H1.1 5 2.2 12.7 I 1 13.4 2.2 31.6 AN 0.54 2.3 1.4 8.4

A chronic model of murine S. pneumoniae lung infection was alsoestablished and the efficacy of a variety of currently availabletetracycline compounds were tested in this model (Table 12).

TABLE 12 Efficacy (PD50) of tetracycline compounds in mice with acutepulmonary infection due to S. pneumoniae PBS1339. PD50 (mg/kg) CompoundIV PO Minocycline 4.5 3.6 Doxycycline 7.1 35.3 Ampicillin ND 3.2 H <5.03.6

EXAMPLE 5 In vivo Murine Model of B. anthracis Infection

In this example, mice were exposed to B. anthracis using whole bodyaerosol challenge, which approximated the mode of pathogen disseminationthat would be expected during a bioterrorist event and was thereforepreferred over other models (e.g., intratracheal inoculation). Animalswere challenged with 75-100 LD50 of B. anthracis Ames strain spores(LD50 3.4×104 CFU/ml), which has been demonstrated repeatedly to causedeath in 90-100% of untreated animals. Treatment with the substitutedtetracycline compounds of the invention began 24 hours after challengeand continued for 21 days. Due to the persistence of ungerminatedanthrax spores in the lungs of challenged animals, a treatment durationof 21 days was used regardless of the antibiotic class. Antibiotics wereadministered by parenteral injection or oral administration. Treatmentgroups were followed for an additional 30 days after cessation ofantibiotic treatment. In addition to monitoring survival in eachtreatment group, animals were sacrificed at selected time points tomonitor microbiological burdens. Tissues, including brain, spleen,lungs, heart, and liver were excised and pathogens were enumerated usingagar plates. Additionally, emergence of resistance was monitored byculturing organs on antibiotic containing media (usually at 3× thebaseline MIC).

Individual treatment groups consisted of 15 animals and the studyendpoint was death after infectious challenge. Ciprofloxacin (30 mg/kg,q12 h) or doxycycline (at experimentally determined doses) was includedas the active control in all experiments. Moribund animals exhibitinglabored breathing, showing signs of paralysis, or that are unresponsivewere humanely euthanized. Challenged survivors were humanely euthanizedat the conclusion of the example.

Throughout the study the mice were observed three to four times dailyand mortality was recorded with each inspection. All moribund mice wereeuthanized and the deaths were recorded as the day of sacrifice. Allmice that died or were sacrificed had their lungs and spleensquantitatively cultured on drug-free and antibiotic supplemented agar(3×MIC) to determine the effect of the treatment regimen on the totaland drug-resistant bacterial populations, respectively.

Twenty-four hours after the last dose was given, a group of survivingmice (n=5) were sacrificed and the lungs and spleens were asepticallyharvested. The homogenized specimens were washed with saline to preventdrug carryover and bacteria were quantitatively cultured on drug-freeand antibiotic-supplemented agar (3×MIC). The remaining animals wereobserved for survival for 14 days after the last dose of drug is given.Those that were alive after the 2-week observation period weresacrificed and their lungs and spleens were quantitatively cultured fortotal and drug-resistant populations. Portions of the homogenates were“heat shocked” for spore determination and bacterial load was determinedby plating onto culture media and incubated at 36° C.

Differences in survival between treatment and control groups wasassessed by the Fisher exact test and by survival analysis techniques(Kaplan-Meier analysis and Cox proportional hazards modeling).Differences in bacterial concentrations in the lungs were determined byStudent's t-test or by ANOVA. A P value <0.05 is consideredstatistically significant.

The results of the in vivo assay indicate that untreated mice exposed toB. anthracis survived approximately 4 days, all mice treated with 10mg/kg compound AN survived the entire 21 days and 75% of mice treatedwith 25 mg/kg of compound AN survived the entire 21 days.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1. A method for treating a bacillus anthracis infection in a subject,comprising administering to said subject an effective amount of asubstituted tetracycline compound, such that said bacillus anthracisinfection in said subject is treated.
 2. The method of claim 1, whereinsaid substituted tetracycline compound has an MIC less than that ofdoxycycline for at least one strain of bacillus anthracis.
 3. The methodof claim 1, wherein said substituted tetracycline compound has an MICless than 32 μg/ml for a doxycycline resistant strain of bacillusanthracis.
 4. The method of claim 1, wherein said substitutedtetracycline compound has an MIC less than that of ciproflaxin for atleast one strain of bacillus anthracis.
 5. The method of claim 1,wherein said substituted tetracycline compound is of the formula I:

wherein R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen, optionally linkedto R² to form a ring; R^(2″) is cyano or C(═O)—NR²R^(2′); R² ishydrogen, alkyl, halogen, alkenyl, alkynyl, aryl, hydroxyl, thiol,cyano, nitro, acyl, formyl, alkoxy, amino, alkylamino, heterocyclic, orabsent, optionally linked to R¹ to form a ring; R^(2′), R³, R^(4a), andR^(4b) are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,heterocyclic, heteroaromatic or a prodrug moiety; R¹⁰, R¹¹, and R¹² areeach independently hydrogen, alkyl, aryl, benzyl, arylalkyl, or apro-drug moiety; R⁴ and R^(4′) are each independently NR^(4a)R^(4b),alkyl, acyl, alkenyl, alkynyl, hydroxyl, halogen, hydrogen, or takentogether ═N—OR^(4a); R⁵ and R^(5′) are each independently hydroxyl,hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy; R⁶ andR^(6′) are each independently hydrogen, methylene, absent, hydroxyl,halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R⁷ ishydrogen, dialkylamino, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, boronic ester, alkylcarbonyl, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a); R⁸ is hydrogen, hydroxyl, halogen, thiol,nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, amino, arylalkenyl, arylalkynyl, acyl,aminoalkyl, heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(8c))₀₋₁C(=E′)ER^(8a); R⁹ is hydrogen, hydroxyl, halogen, thiol,nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, arylalkyl, amino, arylalkenyl, arylalkynyl, acyl,aminoalkyl, heterocyclic, thionitroso, or —(CH₂)₀₋₃ (NR^(9c))₀₋₁C(═Z′)ZR^(9a); R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a),R^(8b), R^(8c), R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d),R^(9e), and R^(9f) are each independently hydrogen, acyl, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety; R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; E is CR^(8d)R^(8e), S, NR^(8b) or O; E′ is O, NR^(8f), or S; W isCR^(7d)R^(7e), S, NR^(7b) or O; W′ is O, NR^(7f), or S; X isCHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O; Y′ and Y are eachindependently hydrogen, halogen, hydroxyl, cyano, sulfhydryl, amino,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl; Z is CR^(9d)R^(9e), S,NR^(9b) or O; Z′ is O, S, or NR^(9f), and pharmaceutically acceptablesalts, esters and enantiomers thereof.
 6. The method of claim 5, whereinR^(2″) is C(═O)NH₂; R³, R¹⁰, R¹¹, and R¹² are each hydrogen or a prodrugmoiety; R⁴ is NR^(4a)R^(4b); R^(4a) and R^(4b) are each methyl; R⁵ ishydrogen; R⁸ is hydrogen; X is CR⁶R^(6′); R⁶ is hydrogen; and R^(5′) andR^(6′) are hydrogen.
 7. The method of claim 6, wherein R⁸ and R⁹ arehydrogen.
 8. The method of claim 7, wherein R⁷ is substituted phenyl, aboronic ester, alkylcarbonyl, heterocyclic, aminoalkyl, or arylalkynyl.9. The method of claim 8, wherein R⁷ is phenyl substituted with alkoxy,alkyl-O—N═C—CR^(7g)R^(7h), alkylaminoalkyl, alkenylaminoalkyl,alkoxyalkylaminoalkyl, substituted alkyl, or substituted carbonylamino,wherein R^(7g) and R^(9h) are each independently hydrogen or alkyl. 10.The method of claim 8, wherein R⁷ is substituted or unsubstitutedheteroaryl.
 11. The method of claim 10, wherein R⁷ is substituted orunsubstituted pyrimidinyl, pyridinyl, or furanyl.
 12. The method ofclaim 8, wherein R⁷ is substituted or unsubstituted piperdinyl-alkyl.13. The method of claim 8, wherein R⁷ is pyridinyl-alkynyl orsubstituted or unsubstituted phenyl alkynyl.
 14. The method of claim 6,wherein R⁷ is hydrogen.
 15. The method of claim 14, wherein R⁹ issubstituted carbonylamino.
 16. The method of claim 6, wherein R⁸ ishydrogen; R⁷ is heterocyclic, alkyl, alkyl-O—N═C—CR^(7g)R^(7h)—, ordimethylamino, wherein R^(7g) and R^(9h) are each independently hydrogenor alkyl.
 17. The method of claim 16, wherein R⁹ is aminoalkyl. 18-36.(canceled)
 37. A pharmaceutical composition comprising an effectiveamount of a substituted tetracycline compound for the treatment of abacillus anthracis infection and a pharmaceutically acceptable carrier.38. The pharmaceutical composition of claim 37, wherein said substitutedtetracycline compound is of formula (I): formula I:

wherein R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen, optionally linkedto R² form a ring; R^(2″) is cyano or C(═O)—NR²R^(2′); R² is hydrogen,alkyl, halogen, alkenyl, alkynyl, aryl, hydroxyl, thiol, cyano, nitro,acyl, formyl, alkoxy, amino, alkylamino, heterocyclic, or absent,optionally linked to R¹ to form a ring; R^(2′), R³, R^(4a), and R^(4b)are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,heterocyclic, heteroaromatic or a prodrug moiety; R¹⁰, R¹¹, and R¹² areeach independently hydrogen, alkyl, aryl, benzyl, arylalkyl, or apro-drug moiety; R⁴ and R^(4′) are each independently NR^(4a)R^(4b),alkyl, acyl, alkenyl, alkynyl, hydroxyl, halogen, hydrogen, or takentogether ═N—OR^(4a); R⁵ and R^(5′) are each independently hydroxyl,hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy; R⁶ andR^(6′) each independently hydrogen, methylene, absent, hydroxyl,halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R⁷ ishydrogen, dialkylamino, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, boronic ester, alkylcarbonyl, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a); R⁸ is hydrogen, hydroxyl, halogen, thiol,nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, amino, arylalkenyl, arylalkynyl, acyl,aminoalkyl, heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(8c))₀₋₁C(=E′)ER^(8a); R⁹ is hydrogen, hydroxyl, halogen, thiol,nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, arylalkyl, amino, arylalkenyl, arylalkynyl, acyl,aminoalkyl heterocyclic, thionitroso, or —(CH₂)₀₋₃ (NR^(9c))₀₋₁C(═Z′)ZR^(9a); R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a),R^(8b), R^(8c), R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d),R^(9e), and R^(9f) are each independently hydrogen, acyl, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety; R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; E is CR^(8d)R^(8e), S, NR^(8b) or O; E′ is O, NR^(8f), or S;W is CR^(7d)R^(7e), S, NR^(7b) or O; W′ is O, NR^(7f), or S; X isCHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O; Y′ and Y are eachindependently hydrogen, halogen, hydroxyl, cyano, sulfhydryl, amino,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl; Z is CR^(9d)R^(9e), S,NR^(9b) or O; Z′ is O, S, or NR^(9f), and pharmaceutically acceptablesalts, esters and enantiomers thereof. 39-49. (canceled)